1. Technical Field
The present invention is in the field of apparatus and method for magnetic cell separation. More particularly, magnetic separation of viable desired cells (target cells) from a heterogeneous cell mixture is accomplished by magnetic separation of desired cells from unwanted cells and other materials while the target cells are bound to paramagnetic beads, forming cell/bead complexes, followed by unbinding of the paramagnetic beads from the target cells, and removal of the paramagnetic beads from the target cells.
2. Related Technology
In the field of cell separation, it is common to separate cells from plasma in blood or bone marrow, and also to separate, by centrifugation, white cells from red cells and platelets. The white cell population separated by standard centrifugation techniques is known as the "buffy coat" fraction. The buffy coat fraction excludes hemoglobin-containing erythrocytic cells as well as platelets. However, the buffy coat fraction does contain a wide mix of hematopoietic mononuclear cells (MNC), including stem cells and progenitors of the erythrocyte, lymphocyte, monocyte, macrophage, granulocyte, and megakaryocyte lineages.
The various types of hematopoietic mononuclear cells are of nearly equal specific gravity, and thus they may not be separated from each other by centrifugation alone. However, different cell types have different sets of cell-surface markers such as specific proteins and glycoproteins on their surfaces. Thus, positive cell separation techniques may exploit selective binding to the desired cell type, also known as the "target cell".
For example, the hematopoietic stem cell is often the desired "target cell" because its progeny have the capacity to differentiate into all the different types of hematopoietic cells. In theory, it is possible to reconstitute a patient's hematopoietic system by infusing a concentrated suspension of stem cells. A stem cell marker known as "CD34 cell surface antigen" may be exploited to separate human hematopoietic stem cells from a mixture of mononuclear cells.
Once positive separation of stem cells becomes practical, new fields of therapy become possible. For instance, a patient's stem cells may be selected, induced to proliferate in an ex vivo culture, and then be returned to the patient after radiation or high dose chemotherapy treatments, which destroy the rapidly-dividing hematopoietic cells of the bone marrow. The selected stem cells may also be induced to differentiate ex vivo to produce more mature cells such as neutrophil and megakaryocyte precursors, which then may be returned to the patient in order to reduce bacterial infections and episodes of bleeding. A concentrated suspension of stem cells would also provide a natural host for gene therapy. For instance, a patient's selected stem cells could be transfected with a gene which, when expressed, could cure a genetic disease of the hematopoietic system such as sickle cell anemia or chronic granulomatous disease. Stem cells also could be transfected with genes which could cure genetic diseases outside of the hematopoietic system. For instance, certain forms of hemophilia could be cured if a small number of the patients' stem cells could be made to produce only a very small amount of certain clotting factors. Also, adjunct cancer therapies are envisioned whereby a patient's stem cells are transfected with a gene which provides resistance to chemotherapeutic agents. After these transfected stem cells repopulate the patient's bone marrow, the patient may be given high-dose chemotherapy for metastatic breast or colon cancer, for instance. The chemotherapeutic drugs will target rapidly dividing metastatic cancer cells, but hematopoietic cells of the bone marrow will be spared because they express a gene which disables the drugs.
The field of cell separation has been divided into two general categories: negative cell separation and positive cell separation. In negative cell separation, the cells that are bound in the device are deleterious cells such as tumor cells that are to be purged from a heterogeneous cell mixture such as blood or bone marrow, and which subsequently is returned to the patient. Thus, in negative separation, it is not critical to maintain the viability of the bound cells. Rather, in negative separation, it is critical to remove 100% of the tumor cells while maintaining the viability of the unbound cells.
Positive cell separation presents special challenges in that it is critical to maintain the viability of the bound cells. One approach to positive cell separation involves the use of an avidin column to which biotinylated secondary antibodies are bound, as may be seen in the following publication, EP 260 280 B1; WO 92/07243; WO 91/16116; WO 91/16088; WO 93/08258.
The heterogeneous cell mixture is first incubated with primary antibodies against CD34, for instance, which specifically binds to stem cells. The cell mixture then flows through the column such that the secondary antibodies on the column bind to the primary antibodies, which in turn are bound to the desired cells. The undesired cells travel through the column with the elution fraction for subsequent disposal. U.S. Pat. No. 5,240,856 (Goffe, et al.), discloses the use of a magnetic stir bar to mix the cell suspension during column processing. The positively selected cells are then mechanically dislodged from the column. However, there are problems associated with this column-based system such as low cell viability, possibly due to mechanical damage; and of low cell purity and yield, possibly due to entrapping of the cells in the column.
Other current practices in the field for cell separation utilize matrix materials of, for example, hollow fibers, flat sheet membrane, or packed-bed bead or particle materials with physically adsorbed or covalently attached chemicals or antibodies for selective cell separation. These devices are designed to allow continuous whole blood or blood component inflow and return. Since these devices operate at normal blood flow rates under conditions in which the concentration of desired cells can be very low compared with other cell types, the separation process is often not efficient. Moreover, with these systems it is difficult to collect the selected cells in a viable state.
The development of paramagnetic beads offered the prospect of magnetic separation of target cells. Various methods to produce magnetic and paramagnetic particles are disclosed in the following United States patents: U.S. Pat. No. 4,672,040 (Josephson); U.S. Pat. No. 5,091,206 (Wong, et al); U.S. Pat. No. 4,177,253 (Davies, et al); U.S. Pat. No. 4,454,234 (Czerlinski); U.S. Pat. No. 4,582,622 (Ikeda, et al); U.S. Pat. No. 4,452,773 (Molday); U.S. Pat. No. 5,076,950 (Ullman); U.S. Pat. No. 4,554,088 (Whitehead); and U.S. Pat. No. 4,695,392 (Whitehead).
Various methods were devised to use magnetic particles for assays. See, for example, United States patents: U.S. Pat. No. 4,272,510 (Smith, et al); U.S. Pat. No. 4,777,145 (Luotola, et al); U.S. Pat. No. 5,158,871 (Rossomando); U.S. Pat. No. 4,628,037 (Chagnon); U.S. Pat. No. 4,751,053 (Dodin); U.S. Pat. No. 4,988,618 (Li, et al); U.S. Pat. No. 5,183,638 (Wakatake); U.S. Pat. No. 4,018,886 (Giaever); and U.S. Pat. No. 4,141,687 (Forrest) .
Attempts were made to use magnetic particles for separation of biological components, including cells. The following is a list of United States patents known to the Applicants and believed to be directed to magnetic separators and methods: U.S. Pat. No. 4,855,045 (Reed); U.S. Pat. No. 4,664,796 (Graham, et al. ); U.S. Pat. No. 4,190,524 (Watson); U.S. Pat. No. 4,738,773 (Muller-Ruchholtz); U.S. Pat. No. 4,941,969 (Schonert); U.S. Pat. No. 5,053,344 (Zhorowski); U.S. Pat. No. 5,200,084 (Liberti); U.S. Pat. No. 4,375,407 (Kronick); U.S. Pat. No. 5,076,914 (Garaschenko); U.S. Pat. No. 4,595,494 (Kukuck); U.S. Pat. No. 4,290,528 (Stekly); U.S. Pat. No. 4,921,597 (Lurie); U.S. Pat. No. 5,108,933 (Liberti, et al.); U.S. Pat. No. 4,219,411 (Yen); U.S. Pat. No. 3,970,518 (Giaever); and U.S. Pat. No. 4,230,685 (Senyei) . All of these devices and methods have met with very limited success due to problems with efficiency of cell separation and retention, and low viability of processed cells.
In attempts to remedy the problems with cell separation, other researchers devised alternate magnetic separator devices, as evidenced by the following United States patents, which proposed, for example, adjustable magnet positions (U.S. Pat. No. 4,710,472; Saur, et al), magnetic gradients (U.S. Pat. No. 4,904,391; Freeman), and magnets of opposite polarity (U.S. Pat. No. 4,910,148; Sorensen, et al). These proposed devices did not solve the two main problems generally encountered in magnetic negative and positive cell separation, i.e., first, the need for very high target cell separation which desirably approaches 100% from a heterogeneous population containing a very low percentage of target cells; and second, the need for removal of nearly all the paramagnetic beads from the final cell product. Thus, these proposed conventional magnetic separator devices did not meet with much success for either negative or positive cell separation.
The central problems for both negative and positive cell separation as described above were solved by utilizing two different magnets in series (Hardwick, et al., Artificial Organs 14:342-347, 1990; co-pending U.S. patent applications Ser. No. 07/979,360 and Ser. No. 07/972,072). In this invention, the first magnet has a relatively strong surface magnetic field strength combined with a broad "reach out" capacity such that its magnetic attractive force acts across the depth of the first container to magnetically attract and bind a high proportion of the paramagnetic particles in the first container. The second magnet in the series has a much stronger magnetic field strength at its surface than the first magnet, such that any paramagnetic beads which escape the first magnet are captured by the second magnet, and thus are removed from the final cell suspension product.
This invention by Hardwick, et al., solved the central problems for cell separation, in general, and for negative cell separation in particular. However, positive cell separation posed an additional challenge in that a greater yield of viable selected cells was desired. Positive cell separation was made practical by inventive changes in the first magnet and in the first container such that desired cells are not crushed as they are drawn on paramagnetic beads to the first magnet (See, "Design of Large-Scale Separation Systems for Positive and Negative Immunomagnetic Selection of Cells Using Superparamagnetic Mircospheres", Hardwick, et al., J. Hematotherapy 1:379-386, 1992, first mailing date to subscribers Jan. 25, 1993). The subject matter of this publication is covered in co-pending United States patent application Ser. No. 08/187,419, filed Jan. 25, 1994 now abandoned. The preferred first magnet for positive cell separation retains certain of the characteristics of the first magnet for negative cell separation in that its "reach out" capacity is sufficiently broad to magnetically attract paramagnetic beads across the depth of the first container. However, the first magnet for positive cell separation has a much lower magnetic field strength at its surface than does the first magnet used for negative cell separation. Moreover, the first container for positive cell separation is noncollapsible. This combination of first magnet and non-collapsible first container permits a much greater yield of viable desired cells. In positive as well as negative cell separation, the second magnet has a much stronger magnetic field strength at its surface than the first magnet.
The system for positive cell separation described immediately above is manually operated, and may be considered somewhat labor intensive. As with all manual systems, the results obtained depend greatly upon the skill of the operator, and may not be as repeatable as is desired. There remains a need for a positive cell separation apparatus which is suitable for a clinical laboratory with limited available labor. Such an apparatus would preferably be semi-automated such that the operator would be free to attend to other duties during most of the cell separation procedure. Also, this semi-automated apparatus would improve the reliability and repeatability of the positive cell separation process by insuring that the operator performs the complex cell separation process correctly each time. Also, the repeatability of the process and quality of results would be improved by such a semi-automated apparatus because the performance of the many steps in the process would be less dependent upon operator skill level, and less affected by the level of the operator's attention to the process.
Accordingly, a primary object for this invention is to provide a semi-automated apparatus for magnetic cell separation.
Another object for the present invention is to provide such an apparatus which includes a semi-automated machine; and a disposable apparatus set in which the cell solution and materials for cell separation are handled and contained during the process. This disposable apparatus set is configured for use with the semi-automated machine, but is fully discarded after one use to avoid contact of laboratory personnel with potentially hazardous biological materials. The single-use disposable set also avoids cross-contamination between patients' blood samples.
Yet another object for the present invention is to provide such a disposable apparatus set in which all of the interconnections of the conduits and chambers of the apparatus set are permanently and fully sealed. This permanent sealing of the apparatus set further insures against contact of laboratory personnel with potentially hazardous biological materials, and improves the purity and sterility of the resulting product of separated target cells.
These and other objects and advantages of the present invention will be apparent from a reading of the following detailed description of a single exemplary preferred embodiment of the invention, taken in conjunction with the appended drawing Figures, in which the same reference numeral refers to the same feature throughout the drawing Figures, or to features which are analogous in structure or function.